Demonstration of Benzo(a)pyrene-Induced DNA Damage in Mice by Alkaline Single Cell Gel Electrophoresis: Evidence for Strand Breaks in Liver but Not in Lymphocytes and Bone Marrow

Abstract: Alkaline single cell gel electrophoresis (also known as the‘comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3×100 mg benzo(a)pyrene/kg b.wt.). No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3×150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.     

Clinicoepidemiological,Toxicological, and Safety Evaluation Studies on Argemone Oil

Abstract

Consumption of oil extracted from accidental or deliberate contamination of argemone seed to mustard seed is known to pose a clinical condition popularly referred to as Epidemic Dropsy. Several outbreaks of Epidemic Dropsy have occurred in the past in India as well as in Mauritius, Fiji Island, and South Africa. Clinico-epidemiological manifestations of argemone oil poisoning include vomiting, diarrhea, nausea, swelling of limbs, erythema, pitting edema, breathlessness, etc. In extreme cases, glaucoma and even death due to cardiac arrest have been encountered. The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Histopathological studies suggest that liver, lungs, kidney, and heart are the target sites for argemone oil intoxication. Studies have shown to elucidate the cocarcinogenic potential of argemone oil that can be correlated with the binding of sanguinarine with a DNA template. Pharmacological response in intestine revealed immediate stimulation of tone and peristaltic movements of the gut in the sanguinarine-treated animals. Argemone oil/Sanguinarine caused a decrease in hepatic glycogen levels which may be due to the activation of glycogenosis leading to an accumulation of pyruvate in the blood of Epidemic Dropsy cases. The increase in pyruvate levels causes uncoupling of oxidative phosphorylation leading to breathlessness, as observed in patients. Sanguinarine has been shown to inhibit Na⁺, K⁺-ATPase activity of different organs such as brain, heart, liver, intestine, and skeletal muscle, which may be due to the interaction with the glycoside receptor site on ATPase enzyme, thereby causing a decrease in the active transport of glucose. Argemone oil/alkaloid showed a Type II binding spectra with hepatic cytochrome P-450 (P-450) protein, thereby causing loss of P-450 content and an impairment of phase I and phase II enzymes. A green fluorescent metabolite of sanguinarine, benzacridine was detected in the milk of grazing animals. The delayed appearance of this metabolite in urine and feces of experimental animals suggests the slow elimination of the alkaloid. Argemone oil enhances hepatic microsomal and mitochondrial lipid peroxidation, indicating that these two organelles are the sites of membrane damage. Furthermore, studies suggest that singlet oxygen and hydroxyl radical are involved in argemone oil toxicity. Several bioantioxidants show protective effect in argemone oil-induced toxicity in experimental animals. The line of treatment in argemone-intoxicated epidemics has so far been only symptomatic, and specific therapeutic measures are still lacking, although it has been suggested that diuretics, bioantioxidants, steroids, vitamins, calcium- and protein-rich diet had some beneficial effects on Epidemic Dropsy cases.     

Detection of Styrene and Styrene Oxide-Induced DNA Damage in Various Organs of Mice Using the Comet Assay

Abstract: Styrene (100–500 mg/kg b. wt.) and styrene oxide (50–200 mg/kg b. wt.) were given as a single intraperitoneal injection to female mice (C57BL/6) at various time intervals before sacrifice. Primary DNA damage in various organs was studied using alkaline single cell gel electrophoresis (comet) assay. Both substances induced significant DNA damage in lymphocytes, liver, bone marrow and kidney after 4 hr. The lymphocytes and liver cells were found to be the most sensitive cells to the DNA damaging effects of both agents. With the exception of bone marrow cells, the degree of DNA damage in all other cell types was decreased from 4 hr to 16 hr after the administration of both compounds. A strong sublinear dose-response relationship was observed in the lymphocytes, liver and bone marrow cells, possibly indicating a saturation of the detoxifying enzyme systems in these organs. The present work suggests that the comet assay can be used for detection of primary DNA damage induced by styrene and styrene oxide in vivo and for comparing the sensitivity of various target organs.     

海牡方对环磷酰胺所致小鼠造血功能损伤的保护作用

目的 研究中药复方制剂海牡方对小鼠造血功能损伤的保护作用。方法 单次尾静脉给予大剂量(250 mg/kg)的环磷酰胺建立小鼠白细胞减少症模型;灌胃给予不同剂量的海牡方受试物,观察对小鼠血细胞、骨髓细胞DNA含量等的影响。结果 海牡方可以促进模型小鼠的白细胞、红细胞及血小板计数水平的恢复(p<0.01),缓解骨髓细胞DNA含量的降低。结论 海牡方能促进环磷酰胺模型小鼠造血机能的恢复,对环磷酰胺所致骨髓损伤具有一定的保护作用。     

The effects in mice of combined treatments to X-rays and antineoplastic drugs in the Comet assay

The Comet assay is a rapid, easy and reproducible method to detect genotoxic activity of chemical and physical agents in vitro and in vivo. In the present study the effects of exposure to irradiation or chemicals: cyclophosphamide (CP) and mitomycin C (MMC) or combined exposure to low doses of both agents (0.25 Gy+3.15 mg/kgbw CP and 0.25 Gy+0.25 mg/kgbw MMC) were examined for the induction of DNA damage in the Comet assay measured simultaneously in somatic (bone marrow lymphocytes) and haploid germ cells. The male mice were treated in vivo and sacrificed at 24 h after exposure. The percentage contents of DNA in the "comet tail" increased with increasing doses of X-rays and chemicals. After combined exposure to X-rays and CP and to X-rays and MMC weak increases of DNA damage in bone marrow lymphocytes and in germ cells were observed by comparison with the results obtained for each agent acting alone. There were slightly different responses in bone marrow lymphocytes and in germ cells, but effects were observed over a similar dose range.     

Skin tumor promotion by argemone oil/alkaloid in mice: Evidence for enhanced cell proliferation,ornithine decarboxylase,cyclooxygenase-2 and activation of MAPK/NF-κB pathway

Consumption of argemone oil (AO) contaminated edible oil causes “Epidemic Dropsy”. Previously, we have shown that AO and isolated sanguinarine possess genotoxicity and skin tumor initiating activity. Here, we evaluate tumor-promoting potential of AO/sanguinarine alkaloid and investigate the molecular mechanisms involved therein. Single topical application of AO (50–400 μl/mouse) or sanguinarine alkaloid (1.5–12.0 μmol/mouse) afforded significant increase in (i) ornithine decarboxylase (ODC) activity, (ii) uptake of [³H]-thymidine in DNA, (iii) cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA) and ODC protein expressions, (iv) phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-jun-N-terminal kinase (JNK)1/2 and p38 mitogen-activated protein (MAP) kinases, (v) increased NF-κB activation and (vi) no significant increase in dark basal keratinocytes. Subsequently, when AO and sanguinarine alkaloid was tested either as complete or stage I or stage II tumor promoter in 7, 12-dimethyl benz(a)anthracene (DMBA)-initiated mice, there was enhanced tumor incidence, tumor body burden and higher % of mice with tumors, when AO (0.1 ml) or isolated sanguinarine (1.5 μmol) was tested as stage II tumor promoter. However, no tumors were found when AO or sanguinarine alkaloid was tested either as complete or stage I tumor promoter. These results indicate that AO/ sanguinarine alkaloid possesses tumor-promoting potential at stage II level involving MAPK/NF-κB pathway.     

Assessing the risk of epidemic dropsy from black salve use

Epidemic dropsy is a potentially life‐threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.     

Flow cytometric detection of apoptotic bone marrow cells with fractional DNA content after application of WR-2721, cyclophosphamide,cisplatin, and exposure of mice to gamma rays

Little is known about the mechanisms of apoptosis triggered in normal cells of the haemopoietic system by the aminothiol WR-2721 (Amifostine), chemotherapeutic drugs, and ionizing radiation; thus, the present study was undertaken to evaluate the effects of WR-2721, cyclophosphamide (CP), cisplatin (CDDP), and 60Co gamma rays on induction of apoptotic DNA degradation in bone marrow cells. Adult male Swiss mice were treated with WR-2721 (400 mg/kg b.wt.), CP (200 mg/kg b.wt.), and CDDP (10 mg/kg b.wt.), and exposed to 6 Gy 60Co gamma rays. Alterations in the number of apoptotic cells with fractional DNA content and also the cell cycle position of the non-apoptotic cells were determined in the bone marrow at 7 and 24 hours after treatment of mice with these agents, using flow cytometric assay of the controlled extraction of low-MW DNA from apoptotic cells. The chemotherapeutic drugs CP and CDDP and 60Co gamma rays triggered apoptosis and affected the cell cycle position of the non-apoptotic cells in the mouse bone marrow. The pretreatment of mice with WR-2721 resulted in the modulatory action of the aminothiol on induction of apoptotic cell death and changes in the cell cycle distribution of the non-apoptotic cells caused by the DNA-damaging agents. The patterns of changes in the frequency of apoptotic cells and the cell cycle position of the non-apoptotic cells, observed in the bone marrow, were dependent on the agent(s) applied and the time interval after application of the drug(s) and exposure of mice to gamma rays. Understanding of the mechanisms responsible for triggering of apoptotic cell death and disturbing of the cell cycle by the DNA-damaging agents, and modulation of the apoptotic and cell cycle pathways by the aminothiol WR-2721, can lead to more effective therapy and chemo- and radio-protection of normal cells.     

螺旋藻多糖对小鼠和犬造血系统的化学和放射防护作用

目的：研究螺旋藻多糖（PSp)对环磷酰胺和^60Co-γ射线所致小白鼠和犬造血系统抑制的影响。方法：腹腔注射环磷酰胺以及用^60Co-γ射线照射分别诱发小鼠和犬的骨髓损伤。全血细胞计数和骨髓有核细胞计数,用紫外分光光度计检测骨髓DNA的含量。结果：环磷酰胺和^60Co-γ射线分别造成小鼠和犬骨髓造血系统抑制性损伤。PSp 30,60mg/kg能升高小鼠全血白细胞数和骨髓有核细胞数以及DNA含量;PSp 12mg/kg能使犬骨髓有核细胞数,以及外周血红细胞、白细胞及血红蛋白水平得以回升（P＜0．01）,其疗效优于盐酸小壁胺。结论：PSp对造血系统有化学保护和放射保护作用。     

Deleterious effects of 28-day oral co-administration of first-line anti-TB drugs on spleen,blood and bone marrow chromosomes in normal rat

Context: Isoniazid, rifampicin and pyrazinamide are most reliable and cost-effective remedy for tuberculosis treatment and prophylaxis among first-line anti-tuberculosis (TB) drugs and have a pronounced tendency to cause adverse drug reactions. Hepatotoxicity is well-studied side effect of these drugs but their effects on other organs like spleen and blood are still needed to be explored. Objective: To explore the probable outcome of co-administration these three major antitubercular drugs (ATDs), rifampicin, isoniazid and pyrazinamide on spleen, blood and bone marrow. Materials and methods: Different parameters were evaluated like lipid peroxidation, glutathione (GSH) and protein content in spleen by spectrophotometric evaluation, hematological evaluation by determining total hemoglobin, total leukocyte count, differential leukocyte count and scanning electron microscopy studies in blood, genotoxicity studied by bone marrow chromosomal analysis and DNA fragmentation. The female rats n?=?12 (150–200?g) were grouped as control group orally given saline and toxicant group given INH (30.85?mg/kg b.wt.)?+?RIF (61.7?mg/kg b.wt.) +?PZA (132.65?mg/kg b.wt.) dosage extrapolated from dose that is used in human for 28 d once daily. Results: After 28 d-oral co-administration of anti-TB drugs (INH (30.85?mg/kg b.wt.)?+?RIF (61.7?mg/kg b.wt.) +?PZA (132.65?mg/kg b.wt.)), it was revealed that there were an increase thiobarbituric acid reactive substances, decrease in GSH and protein contents in spleen. Marked changes in hematological parameters, DNA fragmentation and chromosomes were also observed. Conclusion: This can be concluded from this work that co-administration of first-line ATDs is toxic to spleen and blood also these drugs can cause damage at genetic level.     

In vivo genotoxicity assessment of acrylamide and glycidyl methacrylate

Acrylamide (ACR) and glycidyl methacrylate (GMA) are structurally related compounds used for making polymers with various properties. Both chemicals can be present in food either as a byproduct of processing or a constituent of packaging. We performed a comprehensive evaluation of ACR and GMA genotoxicity in Fisher 344 rats using repeated gavage administrations. Clastogenicity was measured by scoring micronucleated (MN) erythrocytes from peripheral blood, DNA damage in liver, bone marrow and kidneys was measured using the Comet assay, and gene mutation was measured using the red blood cell (RBC) and reticulocyte Pig-a assay. A limited histopathology evaluation was performed in order to determine levels of cytotoxicity. Doses of up to 20 mg/kg/day of ACR and up to 250 mg/kg/day of GMA were used. ACR treatment resulted in DNA damage in the liver, but not in the bone marrow. While ACR was not a clastogen, it was a weak (equivocal) mutagen in the cells of bone marrow. GMA caused DNA damage in the cells of bone marrow, liver and kidney, and induced MN reticulocytes and Pig-a mutant RBCs in a dose-dependent manner. Collectively, our data suggest that both compounds are in vivo genotoxins, but the genotoxicity of ACR is tissue specific.     

Protective effects of indole-3-carbinol on cyclophosphamide-induced clastogenecity in mouse bone marrow cells

Indole-3-carbinol (I3C) is present in many cruciferous vegetables and is known to possess protective properties against chemically induced toxicity and carcinogenesis. In the present study, the antimutagenic potential of I3C has been evaluated using in vivo chromosomal aberration (CA) assay as a cytogenetic end point. Chromosomal analysis was carried out in mouse bone marrow cells following administration of 13C (5 mg/kg; i.p.) for 5 consecutive days. Cyclophosphamide (CP), a well known mutagen, was given at two dose levels of 25 mg/kg b.wt. and 100 mg/kg b.wt., respectively, 24 hours prior to the last dose of I3C. Two groups of five mice each were also injected with CP (25 or 100 mg/kg b.wt.) alone whereas for the vehicle control a group of mice was injected with normal saline only. The results revealed a significant inhibition in the frequencies of CP-induced CAs and aberrant cells in bone marrow cells of I3C-supplemented Swiss albino mice. The antimutagenic potential of 13C towards CP was also evident as the status of mitotic index (MI) was found to show an increment. This study revealed the antigenotoxic potential of I3C against CP-induced chromosomal mutations.     

Evaluation of genotoxicity and DNA protective effects of mangiferin,a glucosylxanthone isolated from Mangifera indica L. stem bark extract

Mangiferin is a glucosylxantone isolated from Mangifera indica L. stem bark. Several studies have shown its pharmacological properties which make it a promising candidate for putative therapeutic use. This study was focused to investigate the in vitro genotoxic effects of mangiferin in the Ames test, SOS Chromotest and Comet assay. The genotoxic effects in bone marrow erythrocytes from NMRI mice orally treated with mangiferin (2000 mg/kg) were also evaluated. Additionally, its potential antimutagenic activity against several mutagens in the Ames test and its effects on CYP1A1 activity were assessed. Mangiferin (50–5000 μg/plate) did not increased the frequency of reverse mutations in the Ames test, nor induced primary DNA damage (5–1000 μg/mL) to Escherichia coli PQ37 cells under the SOS Chromotest. It was observed neither single strand breaks nor alkali-labile sites in blood peripheral lymphocytes or hepatocytes after 1 h exposition to 10–500 μg/mL of mangiferin under the Comet assay. Furthermore, micronucleus studies showed mangiferin neither induced cytotoxic activity nor increased the frequency of micronucleated/binucleated cells in mice bone marrow. In short, mangiferin did not induce cytotoxic or genotoxic effects but it protect against DNA damage which would be associated with its antioxidant properties and its capacity to inhibit CYP enzymes.     

In vivo genotoxic effect of arsenic trioxide in mice using comet assay

Although arsenic has been the subject of toxicological research, acute in vivo genotoxic studies using relevant animal models and uniform methodology are lacking. Hence, the present study aims to study DNA damage caused by arsenic trioxide in mice in in vivo using alkaline single cell gel electrophoresis (Comet) assay. Mice were administered orally 0,0.13,0.27,0.54,1.08,2.15,4.3 and 6.45 mg/kg body weight of arsenic trioxide dissolved in distilled water. The samples of whole blood were collected at 24,48,72 h, first and second week post-treatment and the assay was carried out to determine DNA damage as represented by comet tail-length. All the doses induced significant increase in comet tail-length at 24 h post-treatment (P<0.05) showing a clear dose dependent increase from 0.13 to 2.15 mg/kg b.wt. and a dose dependent decrease in higher doses (4.3-6.45 mg/kg b.wt). At 48 h post-treatment all the doses showed a significant increase (P<0.05) in comet tail-length when compared to 24 h post-treatment. A gradual decrease in the comet tail-length was observed for all the doses from 72 h post-treatment onwards indicating a gradual repair in DNA damage. This indicates a non-linear dose and time response between DNA damage and different doses of arsenic trioxide at different time-intervals. A significant increase in comet tail-length at all the doses clearly gives evidence that arsenic trioxide cause DNA damage effectively. The study indicates that the alkaline comet assay is a reliable and effective method to detect DNA damage caused by metals.     

In vivo genotoxic effects of mercuric chloride in rat peripheral blood leucocytes using comet assay

DNA damage induced by Mercuric chloride (HgCl2) in leucocytes of Wistar albino male rats has been studied in vivo. The comet assay or the alkaline single cell gel electrophoresis (SCGE) assay was used to measure the DNA damage. The rats were administered orally with doses ranging from 0.0054, 0.0108, 0.0216, 0.0432 to 0.0864 mg/kg body weight (b.wt.) of HgCl2. The assay was performed on whole blood at 24, 48, 72 h, 1st and 2nd week. The reason leucocytes were used was to reflect biomarker studies in humans. A significant increase in mean comet tail length indicating DNA damage was observed at all time intervals with HgCl2 except in 2nd week post treatment when compared to controls. The mean comet tail length revealed a clear dose dependent increase from 0.0054 to 0.0432 mg/kg b.wt. A maximum increase in mean comet tail length was observed at 0.0432 mg/kg b.wt. 24 h after treatment. From 48 h post treatment, the mean comet tail lengths of all the doses gradually decreased and by week 2 of post treatment, they had approached control levels, pointing to repair of the damaged DNA. These findings suggest that the comet assay is a highly sensitive technique to study DNA damage caused by metals.     

Genotoxicity testing of low-calorie sweeteners: aspartame, acesulfame-K, and saccharin

Low-calorie sweeteners are chemicals that offer the sweetness of sugar without the calories. Consumers are increasingly concerned about the quality and safety of many products present in the diet, in particular, the use of low-calorie sweeteners, flavorings, colorings, preservatives, and dietary supplements. In the present study, we evaluated the mutagenicity of the three low-calorie sweeteners in the Ames/Salmonella/microsome test and their genotoxic potential by comet assay in the bone marrow cells of mice. Swiss albino mice, Mus musculus, were orally administered with different concentrations of aspartame (ASP; 7, 14, 28, and 35 mg/kg body weight), acesulfame-K (ASK; 150, 300, and 600 mg/kg body weight), and saccharin (50, 100, and 200 mg/kg body weight) individually. Concurrently negative and positive control sets were maintained. The animals were sacrificed and the bone marrow cells were processed for comet assay. The standard plate-incorporation assay was carried with the three sweeteners in Salmonella typhimurium TA 97a and TA 100 strains both in the absence and presence of the S9 mix. The comet parameters of DNA were increased in the bone marrow cells due to the sweetener-induced DNA strand breaks, as revealed by increased comet-tail extent and percent DNA in the tail. ASK and saccharin were found to induce greater DNA damage than ASP. However, none could act as a potential mutagen in the Ames/Salmonella /microsome test. These findings are important, since they represent a potential health risk associated with the exposure to these agents.     

Vetiver oil (Java) attenuates cisplatin-induced oxidative stress,nephrotoxicity and myelosuppression in Swiss albino mice

Clinical efficacy of the widely used anticancer drug cisplatin is limited due to its adverse side effects in normal tissues mediated by oxidative stress. This study was aimed to investigate the protective effects of vetiver acetate oil, Java (VO) against cisplatin-induced toxicity in Swiss albino mice. The ameliorating potential was evaluated by orally priming the animals with VO at doses 5, 10 or 20 mg/kg bw for 7 days prior to cisplatin treatment. Acute toxicity in mice was induced by injecting cisplatin (3 mg/kg bw) intraperitoneally for 5 consecutive days. Significant attenuation of renal toxicity was confirmed by histopathological examination, lowered levels of serum blood urea nitrogen, creatinine and reduced DNA damage. VO also compensated deficits in the renal antioxidant system. VO intervention significantly inhibited DNA damage, clastogenic effects, and cell cycle arrest in the bone marrow cells of mice. Hematological parameters indicated attenuation of cisplatin-induced myelosuppression. Overall, this study provides for the first time that VO has a protective role in the abatement of cisplatin-induced toxicity in mice which may be attributed to its antioxidant activity.     

Protective activity of cedron (Aloysia triphylla) infusion over genetic damage induced by cisplatin evaluated by the comet assay technique

Using the comet assay technique, this paper examines the protection from the cisplatin-induced genetic damage in mouse bone marrow cells provided by cedron-leaf infusion. Animals were separated into six groups: (I) untreated, (II) negative control, (III) treated with cedron-leaf infusion (5%), (IV) treated with cisplatin (6 mg/kg b.w.), (V) pretreated with infusion and treated with cisplatin and (VI) positive control (cyclophosphamide, 20 mg/kg b.w.). Based on the tail moment values found, four types of comets were distinguished. No statistical differences (P<0.01) were found between untreated animals, negative control and infusion treated mice. As expected, treatment of mice with a single dose of cis-DDP-induced genetic damage and the pretreatment with infusion prior to cis-DDP injection inhibited the capacity of cisplatin to induce genetic damage. Cell viability was up to 90% in all cases. The results suggest that infusion could exert its in vivo antigenotoxic action by enhancing the antioxidant status of bone marrow cells. The found could be attributed to its scavenging potency towards free radicals.     

Flow cytometric estimation of the plasma membrane diversity of bone marrow cells in mice treated with WR-2721 and cyclophosphamide

The effects of S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721, Amifostine) and cyclophosphamide (CP) on the cell surface exposure of phosphatidylserine (PS) and the plasma membrane impairment of bone marrow cells were assessed by flow cytometry assay with fluoresceinated annexin V (annexin V - FITC) and propidium iodide (PI). During the 96 h-period after treatment of adult male Swiss mice with WR-2721 (400 mg/kg b.wt.) and CP (200 mg/kg b.wt.), bone marrow cells expressing PS on the outer leaflet of the plasma membrane, which bound annexin V, and cells with a compromised cell membrane, which allowed PI to bind to the cellular DNA, were analysed. Temporary changes in the frequency of early apoptotic cells (annexin V - FITC positive/PI negative), late apoptotic and necrotic cells (annexin V - FITC positive/PI positive), and the number of live cells (annexin V - FITC negative/PI negative), were dependent on the drug(s) given. Application of CP distinctly triggered apoptotic and necrotic cell death, and WR-2721 pre-treatment of mice affected cell death induced by CP, causing reduction of the number of apoptotic and necrotic cells. The chemoprotective action of WR-2721 against PS externalisation and the plasma membrane impairment of normal bone marrow cells was shown.     

Genotoxicity related to transfer of oil spill pollutants from mussels to mammals via food

Heavy fuel oils containing high levels of polycyclic aromatic hydrocarbons (PAHs) were released into the marine environment after the Erika oil spill on the Atlantic coast. As highly condensed PAH pollutants can bioaccumulate in invertebrates, their transfer to vertebrates through the food chain was of concern. This study aimed to estimate potential genotoxic effects in rats fed for 2 or 4 weeks with the marine mussel Mytilus edulis contaminated by oil pollutants. Two levels of PAH contamination were studied, around 100 and 500 microg of total PAHs/kg dry weight (d.w.) in mussels. Genotoxic damage in rats was investigated by single-cell gel electrophoresis (Comet assay) and micronucleus assays in liver, bone marrow, and peripheral blood. DNA damage was observed in the liver of rats fed with the most contaminated mussels (500 microg PAHs/kg d.w.).DNA damage also was observed in the bone marrow but less than that in the liver. A small increase in micronuclei frequency was registered as well. This work underlines the bioavailability of pollutants in fuel-oil-contaminated mussels to consumers and the usefulness of the Comet assay as a sensitive tool in biomonitoring to analyze responses to PAH transfer in food. The occurrence of substituted PAHs and related compounds such as benzothiophenes in addition to nonsubstituted PAHs in fuel oils and mussels raised the question of whether they were implicated in the genotoxic effects registered in rats.